Cs1 targeted chimeric antigen receptor-modified t cells

ABSTRACT

Chimeric antigen receptors for use in treating malignant melanoma and other cancers expressing CS1 are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 15/533,153, filed Jun. 5, 2017, which is a 371 National StageApplication of PCT/US2015/064303, filed Dec. 7, 2015, which claims thebenefit of U.S. Non-Provisional Application No. 62/088,423, filed Dec.5, 2014, entitled “USE OF CENTRAL MEMORY DERIVED-CS1 CHIMERIC ANTIGENRECEPTOR-MODIFIED T CELLS TO TREAT MULTIPLE MYELOMA”, the entirecontents of each of which are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Numbers P50CA107399 and P01 CA030206, awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

Tumor-specific T cell based immunotherapies, including therapiesemploying engineered T cells, have been investigated for anti-tumortreatment. In some cases the T cells used in such therapies do notremain active in vivo for a long enough period. In some cases, theantitumor activity of the T cells is relatively low. Therefore, there isa need in the art for tumor-specific cancer therapies with longer termanti-tumor functioning.

Adoptive T cell therapy (ACT) utilizing chimeric antigen receptor (CAR)engineered T cells may provide a safe and effective way to reducerecurrence rates of various cancers, since CAR T cells can be engineeredto specifically recognize antigenically-distinct tumor populations in anWIC-independent manner.

Multiple myeloma (MM) is a B cell malignancy characterized by clonalexpansion of plasma cells. MM accounts for approximately 1 percent ofall cancers and slightly more than 10 percent of hematologicmalignancies in the United States. In the United States alone,approximately 20,000 new cases will be diagnosed this year and over11,000 people will die from this disease. Current therapies for MM ofteninduce remission, but nearly all patients eventually relapse and die.

CS1 is a cell surface glycoprotein of the signaling lymphocyteactivation molecule (SLAM) receptor family that is highly andselectively expressed on normal plasma cells and MM cells, with lowerexpression on NK cells and little or no expression on normal tissues.Elotuzumabc (HuLuc63), a humanized CS1 monoclonal antibody giventogether with bortezomib in patients with relapsed MM produces ≥PR in48% of patients. The high expression of CS1 on MM cells, coupled withits restriction to plasma cells in normal tissue, makes CS1 a reasonabletarget for CAR T cell therapy (Hsi et al. 2008 Clin Cancer Res 14:2775).

SUMMARY

Described herein are CARs which comprise an extracellular domain, atransmembrane domain and an intracellular signaling domain. Theextracellular domain includes a CS1-specific scFv region or a variantthereof and, optionally, a spacer, comprising, for example, a portion ofhuman Fc domain. The extracellular domain enables the CAR, whenexpressed on the surface of a T cell, to direct T cell activity to cellsexpressing CS1, a receptor expressed on the surface of MM. Thetransmembrane domain includes, for example, a CD4 transmembrane domain,a CD8 transmembrane domain, a CD28 transmembrane domain, or a CD3transmembrane domain. The intracellular signaling domain includes thesignaling domain from the zeta chain of the human CD3 complex (CD3) andone or more costimulatory domains, for example, a 4-1BB costimulatorydomain. The inclusion of a costimulatory domain, such as the 4-1BB(CD137) costimulatory domain in series with CD3 in the intracellularregion enables the T cell to receive co-stimulatory signals. T cells,for example, patient-specific, autologous T cells can be engineered toexpress the CARs described herein, and the engineered cells can beexpanded and used in ACT. Various T cell subsets, including both alphabeta T cells and gamma delta T cells, can be used. In addition, the CARcan be expressed in other immune cells such as NK cells. Where a patientis treated with an immune cell expressing a CAR described herein thecell can be an autologous T cell or an allogenic T cell. In some casesthe cells used are a cell population that includes both CD4+ and CD8+central memory T cells (T_(CM)), which are CD62L+, CCR7+, CD45RO+, andCD45RA−. The cell population can include other types of T cells as well.

The CS1 CAR described herein has certain beneficial characteristics,e.g., persistence and enhanced antitumor activity following adoptivetransfer.

T cells expressing a CAR targeting CS1 can be useful in treatment ofcancers such as MM, as well as other cancers that express CS1. Thus,this disclosure includes methods for treating CS1 expressing cancerusing T cells expressing a CAR described herein.

Described herein is a nucleic acid molecule encoding a CAR comprising: aCS1 scFv (e.g., EVQLVESGGGLVQPGGSLRLSCAASGFDF SRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGSTSGGGSGGGSGGGGSSDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKWYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK; SEQ ID NO:1) or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions); atransmembrane domain selected from: a CD4 transmembrane domain orvariant thereof having 1-5 (e.g., 1 or 2) amino acid modifications(e.g., substitutions), a CD8 transmembrane domain or variant thereofhaving 1-5 (e.g., 1 or 2) amino acid modifications (e.g.,substitutions), a CD28 transmembrane domain or a variant thereof having1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions), and aCD3ζ transmembrane domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); a costimulatory domain(e.g., a CD28 co-stimulatory domain or a variant thereof having 1-5(e.g., 1 or 2) amino acid modifications (e.g., substitutions); or a4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or2) amino acid modifications (e.g., substitutions); or both a CD28co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions) and a 4-1BBco-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications (e.g., substitutions); and a CD3 signalingdomain or a variant thereof having 1-5 (e.g., 1 or 2) amino acidmodifications.

This disclosure also nucleic acid molecules that encode any of the CARsdescribed herein (e.g., vectors that include a nucleic acid sequenceencoding one of the CARs) and isolated T cells that express any of theCARs described herein.

Described herein is a nucleic acid molecule encoding a chimeric antigenreceptor, wherein chimeric antigen receptor comprises: a CS1 scFv; aspacer region; a CD28 or CD4 transmembrane domain, a CD28 costimulatorydomain or a 4-IBB costimulatory domain, an optional GlyGlyGly linker,and a CD3 ζ signaling domain.

In one embodiment, the CS1 CAR consists of or comprises the amino acidsequence of any of SEQ ID NOs:31, 34, 37, 40, 43, and 46 (mature CARlacking a signal sequence) or the CS1 CAR consists of or comprises theamino acid sequence of any of SEQ ID NOs:30, 33, 36, 39, 42, and 45(immature CAR having a GMCSFRa signal sequence). The CAR and can beexpressed in a form that includes a signal sequence, e.g., a humanGM-CSF receptor alpha signal sequence (MLLLVTSLLLCELPHPAFLLIP; SEQ IDNO:26). The CAR can be expressed with additional sequences that areuseful for monitoring expression, for example a T2A skip sequence and atruncated EGFRt. Thus, the CAR can comprise or consist of the amino acidsequence of any of SEQ ID Nos: 29-46 or can comprise or consist of anamino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identicalto any of SEQ ID Nos: 29-46. The CAR can comprise or consist of theamino acid sequence of any of SEQ ID Nos: 29-46 with up to 1, 2, 3, 4 or5 amino acid changes (preferably conservative amino acid changes).

Also disclosed is a population of human T cells transduced by a vectorcomprising an expression cassette encoding a CS1 chimeric antigenreceptor described herein (e.g., a CAR that comprises or consists of theamino acid sequence of any of SEQ ID Nos: 29-46 or an amino acidsequence that is at least 95%, 96%, 97%, 98% or 99% identical to any ofSEQ ID Nos: 29-46 or the amino acid sequence of any of SEQ ID Nos: 29-46with up to 1, 2, 3, 4 or 5 amino acid changes (preferably conservativeamino acid changes).

In various embodiments: the population of human T cells are centralmemory T cells (Tcm), e.g., CD8+/CD4+ Tcm.

An “amino acid modification” refers to an amino acid substitution,insertion, and/or deletion in a protein or peptide sequence. An “aminoacid substitution” or “substitution” refers to replacement of an aminoacid at a particular position in a parent peptide or protein sequencewith another amino acid. A substitution can be made to change an aminoacid in the resulting protein in a non-conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. The following are examples of various groupings of aminoacids: 1) Amino acids with nonpolar R groups: Alanine, Valine, Leucine,Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine; 2) Aminoacids with uncharged polar R groups: Glycine, Serine, Threonine,Cysteine, Tyrosine, Asparagine, Glutamine; 3) Amino acids with chargedpolar R groups (negatively charged at pH 6.0): Aspartic acid, Glutamicacid; 4) Basic amino acids (positively charged at pH 6.0): Lysine,Arginine, Histidine (at pH 6.0). Another grouping may be those aminoacids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.

CS1 ScFv Domain

The CS1 ScFv domain can be any ScFv that binds CS1. In some cases theCS1 ScFv domain includes a sequence that is at least 90%, at least 95%,at least 98% identical to or identical to SEQ ID NO:1. In some cases theCS1 scFv has 1, 2, 3, 4 of 5 amino acid changes (preferablyconservative) compared to SEQ ID NO:1. The ScFv can be a humanized ScFv.

Spacer Region

The CAR described herein can include a spacer region located between theCS1targeting domain (i.e., a CS1 ScFv or variant thereof) and thetransmembrane domain. A variety of different spacers can be used. Someof them include at least portion of a human Fc region, for example ahinge portion of a human Fc region or a CH3 domain or variants thereof.Table 1 below provides various spacers that can be used in the CARsdescribed herein.

TABLE 1 Examples of Spacers Name Length Sequence a3   3 aa AAA linker 10 aa GGGSSGGGSG (SEQ ID NO: 2) IgG4 hinge (S→P)  12 aaESKYGPPCPPCP (SEQ ID NO: 3) (S228P) IgG4 hinge  12 aaESKYGPPCPSCP (SEQ ID NO: 4) IgG4 hinge  22 aa ESKYGPPCPPCPGGGSSGGGSG(S228P) + linker (SEQ ID NO: 5) CD28 hinge  39 aaIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKP (SEQ ID NO: 6) CD8 hinge-48 aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACD (SEQ ID NO: 7)CD8 hinge-45 aa  45 aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 8) IgG4(HL-CH3) 129 aaESKYGPPCPPCPGGGSSGGGSGGQPR (includes S228P EPQVYTLPPSQEEMTKNQVSLTCLVKin hinge) GFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9) IgG4(L235E, 229 aaESKYGPPCPSCPAPEFEGGPSVFLFPPK N297Q) PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFQ STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10) IgG4(S228P, 229 aaESKYGPPCPPCPAPEFEGGPSVFLFPPK L235E, N297Q) PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFQ STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 11) IgG4(CH3) 107 aaGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 12)

Some spacer regions include all or part of an immunoglobulin (e.g.,IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that fallsbetween the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fchinge or a CD8 hinge. Some spacer regions include an immunoglobulin CH3domain or both a CH3 domain and a CH2 domain. The immunoglobulin derivedsequences can include one ore more amino acid modifications, forexample, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduceoff-target binding.

In certain embodiments the spacer is a hinge/linger derived from anIgG1, IgG2, IgG3, or IgG4 that includes one or more amino acid residuessubstituted with an amino acid residue different from that present in anunmodified hinge. The one or more substituted amino acid residues areselected from, but not limited to one or more amino acid residues atpositions 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239,243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 218,326, 330, 331, 332, 333, 334, 336, 339, or a combination thereof.

In some embodiments, the modified hinge of the hinge/liker is derivedfrom an IgG1, IgG2, IgG3, or IgG4 that includes, but is not limited to,one or more of the following amino acid residue substitutions: C220S,C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, L234A, L235A,L235E, G236A, G237A, P238S, S239D, F243L, P247I, S267E, H268Q, S280H,K290S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V,T299A, Y300L, V305I, V309L, E318A, K326A, K326W, K326E, L328F, A330L,A330S, A331S, P331S, 1332E, E333A, E333S, E333S, K334A, A339D, A339Q,P396L, or a combination thereof.

In some embodiments, the modified hinge is derived from a human IgG4hinge/CH2/CH3 region having the amino acid sequence of SEQ ID NO: 10 or11 or an amino acid sequence that is at least 90%, at least 95%, atleast 98% identical to SEQ ID NO:10 or 11.

In certain embodiments, the modified hinge is derived from IgG4 thatincludes one or more amino acid residues substituted with an amino acidresidue different from that present in an unmodified hinge. The one ormore substituted amino acid residues are selected from, but not limitedto one or more amino acid residues at positions 220, 226, 228, 229, 230,233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292,297, 298, 299, 300, 305, 309, 218, 326, 330, 331, 332, 333, 334, 336,339, or a combination thereof.

In some embodiments, the modified hinge is derived from an IgG4 thatincludes, but is not limited to, one or more of the following amino acidresidue substitutions: 220S, 226S, 228P, 229S, 230S, 233P, 234A, 234V,234F, 234A, 235A, 235E, 236A, 237A, 238S, 239D, 243L, 247I, 267E, 268Q,280H, 290S, 290E, 290N, 292P, 297A, 297Q, 298A, 298G, 298D, 298V, 299A,300L, 305I, 309L, 318A, 326A, 326W, 326E, 328F, 330L, 330S, 331S, 331S,332E, 333A, 333S, 333S, 334A, 339D, 339Q, 396L, or a combinationthereof, wherein the amino acid in the unmodified hinge is substitutedwith the above identified amino acids at the indicated position. In oneinstance the sequence includes the following amino acid changes S228P,L235E and N297Q.

For amino acid positions in immunoglobulin discussed herein, numberingis according to the EU index or EU numbering scheme (Kabat et al. 1991Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, herebyentirely incorporated by reference). The EU index or EU index as inKabat or EU numbering scheme refers to the numbering of the EU antibody(Edelman et al. 1969 Proc Natl Acad Sci USA 63:78-85).

The hinge/linker region can also comprise a IgG4 hinge region having thesequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3).

The hinge/linger region can also comprise the sequence ESKYGPPCPPCP (SEQID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2)followed by IgG4 CH3 sequenceGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:12).Thus, the entire linker/spacer region can comprise the sequence:ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:11). In some cases the spacer has 1, 2,3, 4, or 5 single amino acid changes (e.g., conservative changes)compared to SEQ ID NO:11. In some cases, the IgG4 Fc hinge/linker regionthat is mutated at two positions (L235E; N297Q) in a manner that reducesbinding by Fc receptors (FcRs).

Transmembrane Region

A variety of transmembrane domains can be used in the. Table 2 includesexamples of suitable transmembrane domains. Where a spacer region ispresent, the transmembrane domain is located carboxy terminal to thespacer region.

TABLE 2 Examples of Transmembrane Domains Name Accession Length SequenceCD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 13) CD28 NM_00613927 aa FWVLVVVGGVLACYSLLVTVAF IIFWV (SEQ ID NO: 14) CD28(M) NM_00613928 aa MFWVLVVVGGVLACYSLLVTVAF IIFWV (SEQ ID NO: 15) CD4 M35160 22 aaMALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 16) CD8tm NM_001768 21 aaIYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 17) CD8tm2 NM_001768 23 aaIYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 18) CD8tm3 NM_001768 24 aaIYIWAPLAGTCGVLLLSLVITLY C (SEQ ID NO: 19) 41BB NM_001561 27 aaIISFFLALTSTALLFLLFF LTLRFSVV (SEQ ID NO: 20)

Costimulatory Domain

The costimulatory domain can be any domain that is suitable for use witha CD3t signaling domain. In some cases the costimulatory domain is aCD28 costimulatory domain that includes a sequence that is at least 90%,at least 95%, at least 98% identical to or identical to:RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:23; LL to GG aminoacid change double underlined). In some cases the CD28 co-signalingdomain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservativeand preferably not in the underlined GG sequence) compared to SEQ IDNO:23. In some cases the co-signaling domain is a 4-1BB co-signalingdomain that includes a sequence that is at least 90%, at least 95%, atleast 98% identical to or identical to:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some casesthe 4-1BB co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes(preferably conservative) compared to SEQ ID NO:24.

The costimulatory domain(s) are located between the transmembrane domainand the CD3ζ signaling domain. Table 3 includes examples of suitablecostimulatory domains together with the sequence of the CD3ζ signalingdomain.

TABLE 3 CD3ζ Domain and Examples of Costimulatory Domains Name AccessionLength Sequence CD3ζ J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 21) CD28 NM_006139  42 aaRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS (SEQ ID NO: 22) CD28gg*NM_006139  42 aa RSKRSRGGHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRS(SEQ ID NO: 23) 41BB NM_001561  42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24) OX40  42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 25)

In various embodiments: the costimulatory domain is selected from thegroup consisting of: a costimulatory domain depicted in Table 3 or avariant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, aCD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications, a 4-1BB costimulatory domain or a variantthereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2)amino acid modifications. In certain embodiments, a 4-1BB costimulatorydomain or a variant thereof having 1-5 (e.g., 1 or 2) amino acidmodifications in present. In some embodiments there are twocostimulatory domains, for example a CD28 co-stimulatory domain or avariant thereof having 1-5 (e.g., 1 or 2) amino acid modifications(e.g., substitutions) and a 4-1BB co-stimulatory domain or a variantthereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g.,substitutions). In various embodiments the 1-5 (e.g., 1 or 2) amino acidmodification are substitutions. The costimulatory domain is aminoterminal to the CD3ζ signaling domain and in some cases a short linkerconsisting of 2-10, e.g., 3 amino acids (e.g., GGG) is positionedbetween the costimulatory domain and the CD3ζ signaling domain.

CD3ζ Signaling Domain

The CD3ζ Signaling domain can be any domain that is suitable for usewith a CD3ζ signaling domain. In some cases the CD3ζ signaling domainincludes a sequence that is at least 90%, at least 95%, at least 98%identical to or identical to:RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ IDNO:21). In some cases the CD3ζ signaling has 1, 2, 3, 4 of 5 amino acidchanges (preferably conservative) compared to SEQ ID NO:21.

Truncated EGFR

The CD3ζ signaling domain can be followed by a ribosomal skip sequence(e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFRhaving a sequence that is at least 90%, at least 95%, at least 98%identical to or identical to:LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM (SEQ ID NO:28). In some cases the truncated EGFRhas 1, 2, 3, 4 of 5 amino acid changes (preferably conservative)compared to SEQ ID NO:28.

CS1 CAR

The CS1 CAR can include a sequence that is at least 90%, at least 95%,at least 98% identical to or identical to the amino acid sequencedepicted in FIG. 2, FIG. 6, FIG. 7, FIG. 8. FIG. 9 or FIG. 10 (SEQ IDNos: 29-46; either including or excluding the GMCSFRa signal sequenceand either including or excluding the T2A ribosomal skip sequence andthe truncated EGFRt).

Among the CAR targeting CS1 described herein are those summarized inTable 4 in which the spacer region, transmembrane domain andcostimulatory domain(s) for each CAR are indicated.

TABLE 4 Examples of CAR Targeting CS1 SEQ ID Costimulatory Name NO* FIG.Spacer TM Domain(s) CS1scFv-IgG4(HL-CH3)- 29//30//31 2 IgG4(HL-CH3) CD28CD28GG CD28tm-CD28gg-Zeta- T2A-EGFRt. CS1scFv-IgG4(HL-CH3)- 32//33//34 6IgG4(HL-CH3) CD4 4-IBB CD4tm-41BB-Zeta-T2A- EGFRt. CS1qscFv- 35//36//377 IgG4(L235E, N297Q) CD4 4-IBB IgG4(L235E, N297Q)- CD4tm-41BB-Zeta-T2A-EGFRt. CS1scFv-IgG4(L235E, N297Q)- 38//39//40 8 IgG4(L235E, N297Q) CD28CD28GG CD28tm-CD28gg-Zeta- T2A-EGFRt CS1scFv-Linker-CD4tm- 41//42//43 9L CD4 4-IBB 41BB-Zeta-T2A-EGFRt. CS1scFv-Linker- 44//45//46 10 L CD28CD28GG CD28tm-CD28gg-Zeta- T2A-EGFRt *SEQ ID NOs for: entire sequencedepicted including GMCSFRa signal sequence, T2A and EGFRt//sequenceincluding GMCSFRa signal sequence but excluding T2A and EGFRt//sequencefor sequence excluding GMCSFRa signal sequence, T2A and EGFRt.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depiction of a CS1 CAR expressing lentiviralvector (CS1scFv-IgG4(HL-CH3)-CD28gg-Zeta(CO)-T2A-EGFRt_epHIV7). The CS1CAR construct includes: a GMCSF signal sequence, CS1 scFv, IgG4 hingeregion, linker, CH3 domain, a CD28 co-stimulatory domain and CD3ζSignaling domain. The CAR construct is followed by a T2A ribosomal skipsequence, and then suicide gene EGFRt coding sequence. The CAR and EGFRtmolecules are expressed from a single transcript.

FIG. 2 depicts the amino acid sequence of a CS1 CAR that includes signalpeptide, a ribosomal skip sequence and an EGFRt (SEQ ID NO:29).

FIG. 3 is a pair of graphs depicting the results of studies showing thatCS1 CAR re-directed Tcm exhibited cytotoxicity against MM cells.Cytotoxicity of the propagated CS1 CAR T cells was evaluated using4-hour 51Cr release assays after co-culture with 51Cr-labeled targetcells. OKT3 expressing LCLs were used as positive controls since theyengage all TCRs, and CS1-negative AML cells (KG1a) were used as negativecontrols. CS1 CAR, but not un-engineered mock T cells showed specificcytotoxicity against MM cells.

FIG. 4 depicts the results of studies showing that CS1 CAR re-directedTcm cells exhibited effector function in response to stimulation of MMcells. CS1 CAR T cells (10⁵) were co-cultured 6 hours in 96-well tissueculture plates with 10⁵ of MM.1S cells as stimulators. 107adegranulation and intracellular IFNgamma production were analyzed withflow cytrometry. The majority of the CAR T cells identified by Erbituxwere induced to degranulate after engagement with MM cells and IFNgammapositive cells were detected in respond to antigen stimulation.

FIG. 5 depicts the results of studies showing that CS1 CAR re-directedTcm cells eradicate multiple myeloma in vivo. Approximately 2×10⁶Firefly luciferase expressing M1V1.1S cells were inoculated into NSGmice via Intra-tibial injection. 7 days after tumor inoculation, 1×10⁶CS-1 CAR T cells were infused into the tumor bearing mice by intravenousinjection. Tumor burdens were monitored with Xenogen® imaging once aweek. Mice that received un-engineered cells were used as control. CS1CAR T cells completely eradicated MM tumor 14 days post T cell infusion,while un-engineered T cells have no effects on tumor inhibition.

FIG. 6 depicts the amino acid sequence ofCS1scFv-IgG4(HL-CH3)-CD4tm-41BB-Zeta-T2A-EGFRt (SEQ ID NO:32).

FIG. 7 depicts the amino acid sequence ofCS1scFv-IgG4(L235E,N297Q)-CD4tm-41BB-Zeta-T2A-EGFRt (SEQ ID NO:35).

FIG. 8 depicts the amino acid sequence of CS1scFv-IgG4(L235E,N297Q)-CD28tm-CD28gg-Zeta-T2A-EGFRt (SEQ ID NO:38).

FIG. 9 depicts the amino acid sequence ofCS1scFv-Linker-CD4tm-41BB-Zeta-T2A-EGFRt (SEQ ID NO:41).

FIG. 10 depicts the amino acid sequence ofCS1scFv-Linker-CD28tm-CD28gg-Zeta-T2A-EGFRt (SEQ ID NO:44).

FIGS. 11A-11E are the complete nucleotide sequence ofCS1scFv-IgG4(HL-CH3)-CD28gg-Zeta-T2A-EGFRt epHIV7 (SEQ ID NO: 47).

FIG. 12 depicts the results of studies showing that CS1 CAR re-directedTcm cells eradicate multiple myeloma in vivo. 2×106 GFPffluc+ MM.1Scells were inoculated via Intra-tibial injection into NSG mice on day−7. 1×106 central memory T cell (Tcm) derived CS1 CAR+ T cells wereintravenously infused into the tumor bearing mice on day 0. Micereceived no T cells or un-transduced Tcm from the same donor were usedas negative controls. Tumor signals were monitored by biophotonicimaging. Means±SEM of phonton/sec from multiple mice are depicted. TheCAR were those of FIG. 2 (CH2 CD28); FIG. 6 (CH2 4IBB); FIG. 8 (EQCD28); FIG. 7 (EQ 4IBB); FIG. 10 (L CD28) and FIG. 9 (L CD4 IBB).

DETAILED DESCRIPTION

Described below is the structure, construction and characterization ofseveral CS1-specific chimeric antigen receptors (“CAR”). A CAR is arecombinant biomolecule that contains an extracellular recognitiondomain, a transmembrane region, and an intracellular signaling domain.The term “antigen,” therefore, is not limited to molecules that bindantibodies, but to any molecule that can bind specifically to anyreceptor. “Antigen” thus refers to the recognition domain of the CAR.The extracellular recognition domain (also referred to as theextracellular domain or simply by the recognition element which itcontains) comprises a recognition element that specifically binds to amolecule present on the cell surface of a target cell. The transmembraneregion anchors the CAR in the membrane. The intracellular signalingdomain comprises the signaling domain from the zeta chain of the humanCD3 complex and optionally comprises one or more co-stimulatorysignaling domains. CARs can both to bind antigen and transduce T cellactivation, independent of MHC restriction. Thus, CARs are “universal”immunoreceptors which can treat a population of patients withantigen-positive tumors irrespective of their HLA genotype. Adoptiveimmunotherapy using T lymphocytes that express a tumor-specific CAR canbe a powerful therapeutic strategy for the treatment of cancer.

In some cases, the CS1 CAR can be produced using a vector in which theCAR open reading frame is followed by a T2A ribosome skip sequence and atruncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail. Inthis arrangement, co-expression of EGFRt provides an inert,non-immunogenic surface marker that allows for accurate measurement ofgene modified cells, and enables positive selection of gene-modifiedcells, as well as efficient cell tracking of the therapeutic T cells invivo following adoptive transfer. Efficiently controlling proliferationto avoid cytokine storm and off-target toxicity is an important hurdlefor the success of T cell immunotherapy. The EGFRt incorporated in theCS1CAR lentiviral vector can act as suicide gene to ablate the CAR+ Tcells in cases of treatment-related toxicity.

The CAR described herein can be produced by any means known in the art,though preferably it is produced using recombinant DNA techniques.Nucleic acids encoding the several regions of the chimeric receptor canbe prepared and assembled into a complete coding sequence by standardtechniques of molecular cloning known in the art (genomic libraryscreening, overlapping PCR, primer-assisted ligation, site-directedmutagenesis, etc.) as is convenient. The resulting coding region ispreferably inserted into an expression vector and used to transform asuitable expression host cell line, preferably a T lymphocyte cell line,and most preferably an autologous T lymphocyte cell line.

Various T cell subsets isolated from the patient can be transduced witha vector for CAR expression. Central memory T cells are one useful Tcell subset. Central memory T cell can be isolated from peripheral bloodmononuclear cells (PBMC) by selecting for CD45RO+/CD62L+ cells, using,for example, the CliniMACS® device to immunomagnetically select cellsexpressing the desired receptors. The cells enriched for central memoryT cells can be activated with anti-CD3/CD28, transduced with, forexample, a lentiviral vector that directs the expression of an CS1 CARas well as a non-immunogenic surface marker for in vivo detection,ablation, and potential ex vivo selection. The activated/geneticallymodified CS1 central memory T cells can be expanded in vitro withIL-2/IL-15 and then cryopreserved.

Example 1: Construction and Structure of epHIV7 Used for Expression ofCS1-Specific CAR

The pHIV7 plasmid is a parent plasmid from which the clinical vectorsexpressing a CS1 CAR can be derived. The epHIV7 vector used forexpression of the CAR was produced from pHIV7 vector (Wang et al. 2011Blood 118:1255). Importantly, this vector uses the human EF1 promoter todrive expression of the CAR. Both the 5′ and 3′ sequences of the vectorwere derived from pv653RSN as previously derived from the HXBc2provirus. The polypurine tract DNA flap sequences (cPPT) were derivedfrom HIV-1 strain pNL4-3 from the NIH AIDS Reagent Repository.

Construction of pHIV7 was carried out as follows. Briefly, pv653RSN,containing 653 bp from gag-pol plus 5′ and 3′ long-terminal repeats(LTRs) with an intervening SL3-neomycin phosphotransferase gene (Neo),was subcloned into pBluescript, as follows: In Step 1, the sequencesfrom 5′ LTR to rev-responsive element (RRE) made p5′HIV-1 51, and thenthe 5′ LTR was modified by removing sequences upstream of the TATA box,and ligated first to a CMV enhancer and then to the SV40 origin ofreplication (p5′HIV-2). In Step 2, after cloning the 3′ LTR intopBluescript to make p3′HIV-1, a 400-bp deletion in the 3′ LTRenhancer/promoter was made to remove cis-regulatory elements in HIV U3and form p3′HIV-2. In Step 3, fragments isolated from the p5′HIV-3 andp3′HIV-2 were ligated to make pHIV-3. In Step 4, the p3′HIV-2 wasfurther modified by removing extra upstream HIV sequences to generatep3′HIV-3 and a 600-bp BamHI-Sall fragment containing WPRE was added top3′HIV-3 to make the p3′HIV-4. In Step 5, the pHIV-3 RRE was reduced insize by PCR and ligated to a 5′ fragment from pHIV-3 (not shown) and tothe p3′HIV-4, to make pHIV-6. In Step 6, a 190-bp BglII-BamHI fragmentcontaining the cPPT DNA flap sequence from HIV-1 pNL4-3 (55) wasamplified from pNL4-3 and placed between the RRE and the WPRE sequencesin pHIV6 to make pHIV-7. This parent plasmid pHIV7-GFP (GFP, greenfluorescent protein) was used to package the parent vector using afour-plasmid system.

A packaging signal, psi w, is required for efficient packaging of viralgenome into the vector. The RRE and WPRE enhance the RNA transcripttransport and expression of the transgene. The flap sequence, incombination with WPRE, has been demonstrated to enhance the transductionefficiency of lentiviral vector in mammalian cells.

The helper functions, required for production of the viral vector, aredivided into three separate plasmids to reduce the probability ofgeneration of replication competent lentivirus via recombination: 1)pCgp encodes the gag/pol protein required for viral vector assembly; 2)pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence toassist in the transportation of the viral genome for efficientpackaging; and 3) pCMV-G encodes the glycoprotein of thevesiculo-stomatitis virus (VSV), which is required for infectivity ofthe viral vector.

There is minimal DNA sequence homology between the pHIV7 encoded vectorgenome and the helper plasmids. The regions of homology include apackaging signal region of approximately 600 nucleotides, located in thegag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence inall three helper plasmids; and a RRE sequence in the helper plasmidpCgp. It is highly improbable that replication competent recombinantvirus could be generated due to the homology in these regions, as itwould require multiple recombination events. Additionally, any resultingrecombinants would be missing the functional LTR and tat sequencesrequired for lentiviral replication.

The CMV promoter was replaced by the EF1α-HTLV promoter (EF1p), and thenew plasmid was named epHIV7. The EF1p has 563 bp and was introducedinto epHIV7 using NruI and NheI, after the CMV promoter was excised.

The lentiviral genome, excluding gag/pol and rev that are necessary forthe pathogenicity of the wild-type virus and are required for productiveinfection of target cells, has been removed from this system. Inaddition, epHIV7 vector construct does not contain an intact 3′LTRpromoter, so the resulting expressed and reverse transcribed DNAproviral genome in targeted cells will have inactive LTRs. As a resultof this design, no HIV-I derived sequences will be transcribed from theprovirus and only the therapeutic sequences will be expressed from theirrespective promoters. The removal of the LTR promoter activity in theSIN vector is expected to significantly reduce the possibility ofunintentional activation of host genes. Table 5 summarizes the variousregulator elements present in epHIV7.

FIG. 1 is a schematic depiction of CS1 CAR(CS1scFv-IgG4(HL-CH3)-CD28gg-Zeta(CO)-T2A-EGFRt epHIV7), a lentiviralvector containing the CAR construct composed of CS1 scFv, IgG4 hingeregion, linker, a CD28 costimulatory domain and CD3ζ Signaling domain.The CAR construct is followed by a T2A ribosomal skip sequence, and thensuicide gene EGFRt coding sequence. The CAR and EGFRt molecules areexpressed from a single transcript. The entire nucleotide sequence ofthe vector is presented in FIGS. 11A-11E and Table 5 presents positionof various elements of the vector.

TABLE 5 Functional elements of CS1 CAR_epHIV7 Regulatory LocationElements (Nucleotide and Genes Numbers) Comments U5  87-171 5′ Uniquesequence psi 233-345 Packaging signal RRE  957-1289 Rev-responsiveelement flap 1290-1466 Contains polypurine track sequence and centraltermination sequence to facilitate nuclear import of pre-integrationcomplex EF1p Promoter 1524-2067 EF1-alpha Eukaryotic Promoter sequencedriving expression of CD19Rop 2084-4963 Therapeutic insert WPRE5011-5611 Woodchuck hepatitis virus derived regulatory element toenhance viral RNA transportation delU3 5626-5730 3′ U3 with deletion togenerate SIN vector R 5731-5811 Repeat sequence within LTR U5 5812-59253′ U5 sequence in LTR Amp^(R) 6761-7619 Ampicillin-resistance gene CoE1ori 7682-8563 Replication origin of plasmid SV40 ori  8860-=9059Replication origin of SV40 CMV promoter 9073-9672 CMV promoter togenerate viral genome RNA R 9728-86  Repeat sequence within LTR

Example 2: Production of Vectors for Transduction of Patient T Cells

For each plasmid (CS1 CAR_epHIV7; pCgp; pCMV-G; and pCMV-Rev2), a seedbank is generated, which is used to inoculate the fermenter to producesufficient quantities of plasmid DNA. The plasmid DNA is tested foridentity, sterility and endotoxin prior to its use in producinglentiviral vector.

Briefly, cells are expanded from the 293T working cell (WCB), which hasbeen tested to confirm sterility and the absence of viral contamination.A vial of 293T cells from the 293T WCB is thawed. Cells are grown andexpanded until sufficient numbers of cells existed to plate anappropriate number of 10 layer cell factories (CFs) for vectorproduction and cell train maintenance. A single train of cells can beused for production.

The lentiviral vector was produced in sub-batches of up to 10 CFs. Twosubbatches can be produced in the same week leading to the production ofapproximately 20 L of lentiviral supernatant/week. The material producedfrom all sub-batches were pooled during the downstream processing phase,in order to produce one lot of product. 293T cells were plated in CFs in293T medium (DMEM with 10% FBS). Factories were placed in a 37° C.incubator and horizontally leveled in order to get an even distributionof the cells on all the layers of the CF. Two days later, cells weretransfected with the four lentiviral plasmids described above using theCaPO4 method, which involves a mixture of Tris:EDTA, 2M CaCl2, 2×RBS,and the four DNA plasmids. Day 3 after transfection, the supernatantcontaining secreted lentiviral vectors was collected, purified andconcentrated. After the supernatant was removed from the CFs,End-of-Production Cells were collected from each CF. Cells weretrypsinized from each factory and collected by centrifugation. Cellswere resuspended in freezing medium and cryopreserved. These cells werelater used for replication-competent lentivirus (RCL) testing.

To purify and formulate vectors crude supernatant was clarified bymembrane filtration to remove the cell debris. The host cell DNA andresidual plasmid DNA were degraded by endonuclease digestion(Benzonase®). The viral supernatant was clarified of cellular debrisusing a 0.45 μm filter. The clarified supernatant was collected into apre-weighed container into which the Benzonase® is added (finalconcentration 50 U/mL). The endonuclease digestion for residual plasmidDNA and host genomic DNA as performed at 37° C. for 6 h. The initialtangential flow ultrafiltration (TFF) concentration of theendonuclease-treated supernatant was used to remove residual lowmolecular weight components from the crude supernatant, whileconcentrating the virus ˜20 fold. The clarified endonuclease-treatedviral supernatant was circulated through a hollow fiber cartridge with aNMWCO of 500 kD at a flow rate designed to maintain the shear rate at˜4,000 sec-1 or less, while maximizing the flux rate. Diafiltration ofthe nuclease-treated supernatant was initiated during the concentrationprocess to sustain the cartridge performance. An 80% permeatereplacement rate was established, using 4% lactose in PBS as thediafiltration buffer. The viral supernatant was brought to the targetvolume, representing a 20-fold concentration of the crude supernatant,and the diafiltration was continued for 4 additional exchange volumes,with the permeate replacement rate at 100%.

Further concentration of the viral product was accomplished by using ahigh speed centrifugation technique. Each sub-batch of the lentiviruswas pelleted using a Sorvall RC-26 plus centrifuge at 6000 RPM (6,088RCF) at 6° C. for 16-20 h. The viral pellet from each sub-batch was thenreconstituted in a 50 mL volume with 4% lactose in PBS. Thereconstituted pellet in this buffer represents the final formulation forthe virus preparation. The entire vector concentration process resultedin a 200-fold volume reduction, approximately. Following the completionof all of the sub-batches, the material was then placed at −80° C.,while samples from each sub-batch were tested for sterility. Followingconfirmation of sample sterility, the sub-batches were rapidly thawed at37° C. with frequent agitation. The material was then pooled andmanually aliquoted in the Class II Type A/B3 biosafety cabinet in theviral vector suite. A fill configuration of 1 mL of the concentratedlentivirus in sterile USP class 6, externally threaded O-ring cryovialswas used. Center for Applied Technology Development (CATD)'s QualitySystems (QS) at COH released all materials according to the Policies andStandard Operating Procedures for the CBG and in compliance with currentGood Manufacturing Practices (cGMPs).

To ensure the purity of the lentiviral vector preparation, it is testedfor residual host DNA contaminants, and the transfer of residual hostand plasmid DNA. Among other tests, vector identity is evaluated byRT-PCR to ensure that the correct vector is present. All releasecriteria are met for the vector intended for use in this study.

Example 3: Preparation of Tcm Cells Suitable for Use in ACT

T lymphocytes are obtained from a patient by leukopheresis, and theappropriate allogenic or autologous T cell subset, for example, CentralMemory T cells (Tcm), are genetically altered to express the CAR, thenadministered back to the patient by any clinically acceptable means, toachieve anti-cancer therapy.

Tcm that are CD8+ are isolated essentially as described in Wang et al.(J Immunology 35:689, 2012). Briefly, on the day of leukapheresis, PBMCwere isolated by density gradient centrifugation over Ficoll-Paquefollowed by two washes in PBS/EDTA. PBMC were then washed once in PBS,resuspended in X Vivo15 media containing 10% fetal calf serum (FCS),transferred to a 300 cc transfer bag, and stored on a 3-D rotatorovernight at room temperature (RT). The following day, up to 5×10⁹ PBMCwere incubated in a 300 cc transfer bag with clinical grade anti-CD4(2.5 mL), anti-CD14 (1.25 mL), and anti-CD45RA (2.5 mL) microbeads(Miltenyi Biotec) for 30 minutes at RT in X Vivo15 containing 10% FCS.CD4+, CD14+ and CD45RA+ cells were then immediately depleted using theCliniMACS™ depletion mode according to the manufacturer's instructions(Miltenyi Biotec). After centrifugation, the unlabeled negative fractionof cells was resuspended in CliniMACS™ PBS/EDTA buffer (Miltenyi Biotec)containing 0.5% human serum albumin (HSA) and then labeled with clinicalgrade biotinylated-DREG56 mAb (COHNMC CBG) at 0.1 mg/106 cells for 30minutes at RT. The cells were then washed and resuspended in a finalvolume of 100 mL CliniMACS™ PBS/EDTA containing 0.5% HSA and transferredinto a new 300 cc transfer bag. After 30 minutes incubation with 1.25 mLanti-biotin microbeads (Miltenyi Biotec), the CD62L+ fraction of PBMC(CD8+ TCM) was purified with positive selection on CliniMACS™ accordingto the manufacturer's instructions, and resuspended in X Vivo15containing 10% FCS.

Tcm that are CD8+/CD4+ are prepared using a modification of the forgoingprocess by modifying the CD4+, CD14+ and CD45RA+ selection to a CD14+and CD45RA+ selection. The method uses a two-step process on theCliniMACS™ device to first deplete CD14+ and CD45RA+ cells, then topositively select CD62L+ cells. This modified platform generates 50×10⁶bulk Tcm from a single leukapheresis.

Following enrichment, Tcm cells are formulated in complete X-Vivo15 plus50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cellculture bag, where they are stimulated with Dynal ClinEx™ Vivo CD3/CD28beads. Up to five days after stimulation, cells are transduced withlentiviral vector encoding CS1 CAR at a multiplicity of infection (MOI)of about 3. Cultures are maintained for up to 42 days with addition ofcomplete X-Vivo15 and IL-2 and IL-15 cytokine as required for cellexpansion (keeping cell density between 3×10⁵ and 2×10⁶ viable cells/mL,and cytokine supplementation every Monday, Wednesday and Friday ofculture). Cells typically expand to approximately 10⁹ cells under theseconditions within 21 days. At the end of the culture period cells areharvested, washed twice and formulated in clinical gradecryopreservation medium.

On the day(s) of T cell infusion, the cryopreserved and released productwill be thawed, washed and formulated for re-infusion. The cryopreservedvials containing the released cell product will be removed from liquidnitrogen storage, thawed, cooled and washed with a PBS/2% human serumalbumin (HSA) Wash Buffer. After centrifugation, the supernatant will beremoved and the cells resuspended in a Preservative-Free Normal Saline(PFNS)/2% HSA infusion diluent. Samples will be removed for qualitycontrol testing.

Example 4: Amino acid Sequence of CS1 CAR(CS1scFv-IgG4(HL-CH3)-CD28tm-CD28gg-Zeta-T2A-EGFRt)

The complete amino acid sequence ofCS1scFv-IgG4(HL-CH3)-CD28tm-CD28gg-Zeta-T2A-EGFRt is depicted in FIG. 2.The entire sequence (SEQ ID NO:29) includes: a 22 amino acid GMCSFsignal peptide (SEQ ID NO:26), a CS1 scFv sequence (SEQ ID NO:1); a IgG4hinge sequence (SEQ ID NO:3; with amino acid substitutions S to Pshaded); a 10 amino acid linker (SEQ ID NO:2); IgG4 CH3 sequence (SEQ IDNO:12); a 28 amino acid CD28 transmembrane domain sequence (SEQ IDNO:14); a CD28gg co-stimulatory domain sequence (SEQ ID NO:23; LL to GGamino acid changes highlighted); a 3 amino acid Gly linker; a 112 aminoacid CD3ζ sequence (SEQ ID NO:21); a 24 amino acid T2A skip sequence(SEQ ID NO:27); and EGFRt sequence (SEQ ID NO:28).

Example 5: Activity of CS1 CAR

Cytotoxicity of the propagated CS1 CAR T cells expressing the CAR shownin FIG. 2 was evaluated using 4-hour 51Cr release assays afterco-culture with 51Cr-labeled MM cells (MM.1S). As shown in FIG. 3, theengineered CS1 CART cells exhibit specific and efficient killing of MMcells, while un-transduced mock T cells has no cytocoxicity to MM cells.When co-cultured with MM cells, the engineered CS1 CAR Tcm-mediatedstrong effector function as indicated by 107a degranulation and IFNgammaas shown in FIG. 4. Upon adoptively transferred into MM tumor bearingNSG mice, the CS1 specific T cells exhibited efficient antitumoractivity as shown in FIG. 5.

In another study with additional CS1 CAR (FIG. 2 and FIGS. 6-10) 2×10⁶GFPffluc+MM.1S cells were inoculated via Intra-tibial injection into NSGmice on day −7.1×10⁶ central memory T cell (Tcm) derived CS1 CAR+ Tcells were intravenously infused into the tumor bearing mice on day 0.Mice received no T cells or un-transduced Tcm from the same donor wereused as negative controls. Tumor signals were monitored by biophotonicimaging. Means±SEM of phonton/sec from multiple mice are depicted. Theresults of this analysis are shown in FIG. 12.

1. A chimeric antigen receptor (CAR) or polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 38, 39, and
 40. 2. The CAR or polypeptide of claim 1, wherein the chimeric antigen receptor or polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 29, 30, and
 31. 3. The CAR or polypeptide of claim 1, wherein the chimeric antigen receptor or polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 32, 33, and
 34. 4. The CAR or polypeptide of claim 1, wherein the chimeric antigen receptor or polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 38, 39, and
 40. 5.-55. (canceled)
 56. The population of human T cells expressing a chimeric antigen receptor (CAR) or polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 38, 39, and
 40. 57.-64. (canceled)
 65. The population of human T cells of claim 56, wherein at least 20%, 30%, 40%, 50%, 60%, 70% or 80% of the human T cells are central memory T cells.
 66. The population of human T cells of claim 56, wherein at least 10% or 20% of the central memory T cells are CD4+.
 67. The population of human T cells of claim 56, wherein at least 10% or 20% of the central memory T cells are CD8+.
 68. The population of human T cells of claim 56, wherein at least 10% of the central memory T cells are CD4+ and at least 10% are CD8+.
 69. (canceled)
 70. A method of treating cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising the human T cells of claim
 56. 71. The method of claim 70 wherein the population of human T cells are autologous to the patient.
 72. The method of claim 70 wherein the population of human T cells are allogenic to the patient.
 73. The method of claim 70 wherein the human T cells are prepared by a method comprising obtaining T cells from the patient or obtaining T cells allogenic to the patient, treating the obtained T cells to isolate a population of cells enriched for CD4+/CD8+ central memory T cells, and transducing at least a portion of the isolated population of cells to with a viral vector comprising an expression cassette encoding a chimeric antigen receptor or polypeptide, wherein chimeric antigen receptor or polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 38, 39, and
 40. 74.-79. (canceled)
 80. The method of claim 70, wherein the cancer is multiple myeloma. 